The number of circulating cells in the blood of patients is a routinely used biomarker in clinical diagnostics. However, the current systems used to detect them are bulky, expensive, time-consuming and require external sample preparation procedures. In addition, microfluidic devices have been utilized, providing compact and cheap solutions that may offer promise to move the testing towards the patient in a bedside setting. Unfortunately, these techniques often require a certain amount of sample preparation, especially when using label-free approaches to achieve a good efficiency.
Recent developments have included using specific sub-populations of cells as biomarkers for various diseases. Sub-types of circulating cells have been used as diagnostic biomarkers for various conditions, for example circulating tumour cells (CTCs) for cancer (Mocellin S. et al., “Circulating tumor cells: the ‘leukemic phase’ of solid cancers”, TRENDS in Molecular Medicine, 2006, 12 (3), 130-139), lymphocytes CD4 for HIV (Jokerst J V. et al., “Integration of semiconductor quantum dots into nano-bio-chip systems for enumeration of CD4+ T cell counts at the point-of-need”, Lab Chip, 2008, 8, 2079-2090) and endothelial progenitor cells for cardiovascular conditions (Massa M. et al., “Increased circulating hematopoietic and endothelial progenitor cells (EPCs) in the early phase of acute myocardial infarction”, Blood, 2005, 105: 199-206). These biomarkers can be used for early diagnostics, prognosis, therapy monitoring or minimal residual disease controls. Their numbers in blood can vary from extremely low, for example <5 cells per 7.5 ml for CTCs, to relatively abundant, for example 200 cells/μl for CD4 T-lymphocytes, with EPCs around 0.01%-2% of peripheral blood mononuclear cells (PBMCs), while blood contains about 7000-10000 white blood cells and 1000 times more red blood cells. This poses a great challenge for biosensor applications requiring very low detection limits in order to detect the biomarkers in the blood.
The number of circulating cells has been used as a diagnosis marker in conventional procedures, such as the white blood cell counts which are performed routinely in the clinical labs. EPCs have also been used as biomarkers of cardiovascular conditions. Circulating EPCs are stem cells derived from the bone marrow with the ability to differentiate into vascular endothelial cell for blood vessel lining repair and their number in blood may be used as a biomarker (Rosenzweig A., “Circulating Endothelial Progenitors—Cells as Biomarkers”, The New England Journal of Medicine, 2005, 353 (10), 1055-1057; J. M. Hill et al., “Circulating Endothelial Progenitor Cells, Vascular Function, and Cardiovascular Risk”, The New England Journal of Medicine, 2003, 348, 593-600; P. E. Szmitko et al., “Endothelial progenitor cells: new hope for a broken heart”, Circulation, 2003, 107, 3093-100). EPC levels may be used for health monitoring as their levels in blood correlate to the coronary artery diseases or cardiovascular conditions and their risk factors. EPCs are also used for therapy monitoring to monitor the effects of primary and secondary prevention strategies, where specific drugs, such as statins, are known to increase the EPC counts. In addition, EPCs can be transplanted for tissue regeneration.
However, analyzing specific subtypes of circulating cells is not a trivial matter and has not been used as a common practice due to technical limitations and relatively high costs. Effective and selective extraction of rare target cells from whole blood has been very challenging for the micro total analysis systems (μTAS). A 1 μl whole blood sample may contain approximately 4-5 millions of red blood cells (RBCs) and approximately 4-11 thousands of peripheral blood mononuclear cells (PBMCs). Assuming detection of CD34+ cells at the level of 0.1% PBMCs, this will imply as few as 7 cells in 1 μl of whole blood. Conventionally, a sample preparation assay for cell purification is required in order to separate such a low concentration of EPCs from blood. Typical procedures include: (1) incubate sample with RBC lysis buffer, (2) centrifuge the cell suspension and remove the supernatants, (3) label with magnetic beads which are tagged with antigen-specific antibodies and (4) centrifuge again and remove unbound beads in the solution. The overall time for the sample preparation process may be around 1-2 hours and in addition, the process requires a bulky centrifuge machine and skilled personnel. Consequently, these present limitations for the use of conventional sample purification assay for point-of-care applications.
The cell sub-types are usually defined by their expressions of specific surface markers. For example, the detection of CTCs is generally based on the presence of the specific epithelial marker, epithelial cell adhesion molecule (EpCAM), on their surface, while EPCs can be defined by its CD34 or CD133 protein or the endothelial marker protein, VEGFR2/KDR or a combination of these proteins. In order to detect these specific cells, the conventional technique is the flow cytometry analysis (Khan S. S. et al., “Detection of Circulating Endothelial Cells and Endothelial Progenitor Cells by Flow Cytometry”, Clinical Cytometry, 2005, 64B, 1-8), such as the fluorescent cell sorter (FACS) which optically reads the fluorescence of cells stained with a specific marker passing through a thin capillary. However, this technique is cumbersome, time-consuming (approximately 4-5 hours for the staining process and analysis), require large sample volumes (>1 ml), demands highly skilled personnel and is generally performed off-site.
With the advent of microfluidics, approaches have emerged which aim to overcome the disadvantages of the FACS (ie. the time and skill involved) and enhance portability. Flow-through systems (Taek Dong Chung, Hee Chan Kim, “Recent advances in miniaturized microfluidic flow cytometry for clinical use”, Electrophoresis, 2007, 28, 4511-4520) directly miniaturize the sorting concept and use specific properties of the cells to direct them to counters, for detection by means of either optical or label-free (Roeser T. et al., “Lab-on-chip for the Isolation and Characterization of Circulating Tumor Cells”, Proceedings of the 29th Annual International Conference of the IEEE EMBS, 2007, 6446-6448), or both (Wang Y-N et al, “On-chip counting the number and the percentage of CD4+ T lymphocytes”, Lab Chip, 2008, 8, 309-315). These systems enable precise counting of the cells passing through, but require preliminary off-chip sample preparation, for example involving fluorescent or magnetic staining, and which may also include separating the PBMCs. Furthermore, flow-through systems may result in cell loss and may not be suitable for large sample processing, which will affect costs and sensitivity/specificity issues. The process of labeling or staining the cells is also time-consuming.
“Flow-stop” systems are also available, which use the specific binding of the cells on the functionalized surfaces of the microdevices to purify the sample directly on the chip from whole blood (Nagrath S. et al. “Isolation of rare circulating tumour cells in cancer patients by microchip technology”, Nature, 2007, 450, 1235-1239). However, detection is performed optically after fluorescent staining and requires a complicated optical analysis system to automate, and with a relatively poor efficiency.
The use of additional labels has prevented the conventional devices from achieving point-of-care detection in a portable manner, with a speed that is amenable for diagnosis of acute diseases, for example <1 hour for acute cardiovascular conditions. Chamber systems have coupled label-free detection with surface specific cell selection to avoid the use of labels at the detection stage. Most of these systems rely on samples, such as PBMCs, that are pre-purified (Ng S Y et al., “Label-free Impedance Detection of Low Levels of Circulating Endothelial Progenitor Cells for Point-of-Care Diagnosis”, Biosensors and Bioelectronics, 2010, 25, 1095-1101). However, their reliance on surface specific capture is limiting, since the much more abundant red blood cells can mask the access of the surfaces for the other cells.
Conventional preparation methods for PBMCs are based on the different in size and density in comparison to their counterparts, such as the red blood cells. For cell-based detection, it is not necessary to separate the other constituents (such as plasma) from the cells, although the techniques used usually do.
There are a number of conventional methods for preparing PBMCs, with the most popular method being centrifugation where the cells are recovered in a buffy coat layer in specific tubes containing different density portions after the centrifugation procedure. The blood samples required are usually in the order of milliliters (ml), and are drawn by syringes and require lab facilities for preparation. Therefore, the preparation of PBMCs off-chip drastically diminishes the interest of using microfluidic devices for point-of-care applications, which need to handle small volumes (for example a blood sample from a finger prick is about 50 μl), at the patient's side.
The concept of centrifugation has also been used on chips to provide pumping of samples through channels and chambers. It is also applied to the separation of cells and other blood constituents (Kang D-R et al., “Blood micro-separator”, US2006/0263265), but requires a rotation mechanism and complex integration schemes for detection.
Size filtration using porous membranes, either microfabricated (Siyang Zheng et al., “Membrane microfilter device for selective capture, electrolysis and genomic analysis of human circulating tumor cells” Journal of Chromatography A, 2007, 1162, 154-161) or paper-like (Vona G. et al., “Isolation by Size of Epithelial Tumor Cells”, American Journal of Pathology, 2000, 156 (1), 57-63; Illert W., “Methods of preparing peripheral stem cells from leukocytes reduction filters”, EP1484390), has been used for the preparation and detection of rare circulating cells (mostly CTCs). In these systems, the sample is passed through a membrane containing pores of specific sizes that will let the small cells (eg. red blood cells) go through, while retaining bigger cells (eg. CTCs and/or white blood cells). The efficiencies achieved are relatively high and the cells are analyzed directly on the membranes, either by optical or biomolecular inspection. However, such techniques cannot be easily integrated in a label-free point-of-care system, which require counting of cells on the membranes or transferring the lysed samples to a specific detector, such as a real-time PCR machine, which does not provide accurate levels.
Size filters have also been microfabricated in completely sealed systems (Maltezos G. et al., “Fluorescence detector, filter device and related methods”, US2008/0013092; Battrell C. F. et al., “Method and system for microfluidic manipulation, amplification and analysis of fluids, for example, bacteria assays and antiglobulin testing”, U.S. Pat. No. 7,416,892). However, the systems are usually used to trap big unwanted particles and detect small (molecules) species. Another drawback of these systems lies in the planar fabrication technologies (mainly silicon processes) which drastically reduce the area of trapping in the systems.